Extracellular Matrix Composition Reveals Complex and ......Extracellular Matrix Composition Reveals...

Extracellular Matrix Composition Reveals Complexand Dynamic Stromal-Epithelial Interactionsin the Mammary Gland

Ori Maller & Holly Martinson & Pepper Schedin

Received: 30 July 2010 /Accepted: 16 August 2010 /Published online: 2 September 2010# Springer Science+Business Media, LLC 2010

Abstract The mammary gland is an excellent modelsystem to study the interplay between stroma and epithelialcells because of the gland’s unique postnatal developmentand its distinct functional states. This review focuses on thecontribution of the extracellular matrix (ECM) to stromal-epithelial interactions in the mammary gland. We describehow ECM physical properties, protein composition, andproteolytic state impact mammary gland architecture aswell as provide instructive cues that influence the functionof mammary epithelial cells during pubertal gland devel-opment and throughout adulthood. Further, based on recentproteomic analyses of mammary ECM, we describe knownmammary ECM proteins and their potential functions, aswell as describe several ECM proteins not previouslyrecognized in this organ. ECM proteins are discussed in

the context of the morphologically-distinct stromal sub-compartments: the basal lamina, the intra- and interlobularstroma, and the fibrous connective tissue. Future studiesaimed at in-depth qualitative and quantitative characteriza-tion of mammary ECM within these various subcompart-ments is required to better elucidate the function of ECM innormal as well as in pathological breast tissue.

Keywords Extracellular matrix . Stromal-epithelialinteractions . Basal lamina . Collagen . Fibronectin .

Laminin

Abbreviationscollagen IV type IV collagenDDR1 discoidin domain receptor 1EM electron microscopyEGFR epidermal growth factor receptorECM extracellular matrixED extra domainFACIT fibril associated collagens with

interrupted triple helicesFGF fibroblast growth factorFN fibronectinGAG glycosaminoglycanH&E hematoxylin and eosin stainHGF hepatocyte growth factorLN lamininLAP latency-associated peptideLRR leucine-rich repeatsLOX lysyl oxidaseMMP matrix metalloproteinaseMAGP microfibril-associated glycoproteinMCP-1 monocyte chemoattractant protein-1SPARC secreted protein acidic and rich in cysteineSLRP small leucine-rich proteoglycan

Financial Support Supported by Department of Defense IdeaAward#BC095850 to PS.

O. Maller :H. Martinson : P. Schedin (*)Department of Medicine, Division of Medical Oncology,University of Colorado Denver,MS8117, RC-1S, 8401K, 12801 E 17th Ave,Aurora, CO 80045, USAe-mail: [email protected]

O. Maller :H. Martinson : P. SchedinProgram in Cancer Biology, University of Colorado Denver,MS8104, RC-1S, 5117, 12801 E 17th Ave,Aurora, CO 80045, USA

P. SchedinUniversity of Colorado Cancer Center,Bldg 500, Suite 6004C, 13001 E 17th Place,Aurora, CO 80045, USA

P. SchedinAMC Cancer Research Center,Bldg 500, Suite 6004C, 13001 E 17th Place,Aurora, CO 80045, USA

J Mammary Gland Biol Neoplasia (2010) 15:301–318DOI 10.1007/s10911-010-9189-6

SD Sprague DawleyTN tenascintTG2 tissue transglutaminase 2TGF-β transforming growth factor βTLR toll like receptor

Introduction

The mammary gland is an excellent model system to studythe function of stromal-epithelial interactions because of thegland’s unique development across its several distinctfunctional states, depending on reproductive state. Unlikemost organs, which develop to morphological maturityduring embryogenesis, the majority of mammary ductalmorphogenesis occurs with the onset of ovarian function.Further, with each estrous (rodent) or menstrual (human)cycle, the alveoli undergo cyclic expansion and maturation,followed by a modest regression phase as ovarian hormonelevels rise and fall, respectively [1–4]. The mammary glandis also unique in that terminal differentiation does not occurunless pregnancy and lactation ensue [5]. These uniqueattributes highlight the dynamic, non-homeostatic nature ofthe normal mammary gland.

Since mammary gland development is largely postnatal,organogenesis and differentiation are extended in time,occurring over a period of weeks in the rodent, rather thanhours and days as observed in other organs. Further, inrodents, the development of mammary glands occurs alongthe entire flank of the animal with 5 pairs of glands in themouse and 6 pairs in the rat. As a result, there is sufficientmammary tissue in these species to permit physical,functional, and biochemical characterizations. These fea-tures make the rodent mammary gland a highly suitablemodel to evaluate the interplay between stroma andepithelium throughout gland development and duringdistinct functional states such as pregnancy or lactation.

The focus of this review is on the extracellular matrix(ECM) component of the mammary stroma. ECM can bedescribed as an interconnected meshwork of secretedproteins that interacts with cells to form a functional unit[6]. Cell-ECM interactions have been implicated in celladhesion, survival, apoptosis, polarity, proliferation, anddifferentiation. ECM confers these cellular functions throughstructural properties such as physical support and boundaryconstraints, classical signal transduction pathways, and byproviding mechanosensory cues [7]. Epithelial cells receiveinstructive cues from the ECM via integrin receptors andthe non-integrin receptors such as discoidin domainreceptor 1 (DDR1) [8], dystroglycan [9], and syndecan[10]. Integrins are transmembrane receptors composed of αand β subunits, with 18 α and 8 β subunits that

heterodimerize to generate 24 canonical integrin receptors[11, 12]. A comprehensive description of ECM-integrininteractions is beyond the scope of this review; the reader isreferred to reviews by Desgrosellier et al. and Larsen et al.[13, 14].

ECM synthesis and assembly in the mammary glandare the product of many cell types including epithelialcells, myoepithelial cells, fibroblasts, adipocytes, endo-thelial cells, and immune cells [15–17]. Bissell andcolleagues describe the relationship between ECM andmammary epithelial cells as dynamic and reciprocal,leading to two-way communication, where ECM instructsand supports cells, while cells build, shape, and re-shapethe ECM [18]. Mammary ECM can be separated into threebroad structural categories: the highly-specialized basallamina that directly abuts the basal side of mammaryepithelial cells (Fig. 1), the intra- and interlobular stromaimmediately adjacent to alveoli and lobules, respectively(Figs. 1 and 2), and the fibrous connective tissue that isdevoid of epithelium (Fig. 2). In this review we explorethe composition and function of each of these ECMcategories using data obtained largely from rodent models.Although rodent models recapitulate many aspects ofhuman mammary gland biology, there are interspeciesvariations in mammary stroma composition, organization,density, and function. Thus, while significant insight intomammary ECM is obtained through the study of rodents,future studies validating results in human breast tissue areessential.

Rodent Mammary Gland as a Model System to StudyHuman Breast

The mouse and rat models recapitulate critical aspects ofhuman mammary gland development and function. Therodent mammary gland and human breast have similarrudimentary mammary parenchyma, which consist of alayer of luminal epithelial cells, a basal epithelial cell layerthat putatively contains mammary stem cells, and the outercontractile myoepithelial cells adjacent to the basal laminaECM [19]. The parenchyma structure consists of a singleelongated ductal tree (rodent) or multiple ductal networks(human) that develop by bifurcation of specialized end budstructures present during puberty [20]. While the overallepithelial structure is similar between humans, rats, andmice, the relative abundance of connective tissue variesconsiderably. Stroma surrounding the lobules and ducts(intra- and interlobular stroma) in mice is sparse and thereis little acellular fibrous connective tissue between ducts.Instead, the mammary glands of mice are characterized byan abundance of white adipose tissue that is directlyadjacent to the sparse interlobular stroma (Fig. 2c). In

302 J Mammary Gland Biol Neoplasia (2010) 15:301–318

humans the ratio of fibrous connective tissue to adiposetissue is opposite, with an abundance of stroma surroundingthe alveoli and ducts, a predominance of fibrous connectivetissue between ducts, and reduced adipose content (Fig. 2a).Organization of the stroma in outbred Sprague Dawley(SD) female rats is intermediate between mice and humansand thus is histologically more similar to humans than is themouse (Fig. 2b) [21]. While the mechanisms driving thedifferent ratios of fibrous connective to adipose tissuesacross species are currently unknown, we suggest arelationship to ovarian hormone exposure, particularlyprogesterone, may exist. Compared to mice, the SD rathas a robust luteal phase during the estrus cycle, resulting incyclical progesterone exposure that is more similar to thatfor women [1]. Given that pituitary and ovarian hormoneexposure drives most aspects of gland development andadult function, instructive cues from hormones are expectedto also directly or indirectly influence composition andorganization of the adjacent stroma [22–24]. Hence,studying the hormonal regulation of normal mammarygland biology may afford novel insights into how stromalorganization is regulated under physiological conditions,which can be utilized to decipher this relationship in termsof tissue plasticity and breast disease. However, systemic/

host exposures are unlikely to completely explain variationin stromal density given that very distinct microenviron-ments can exist within individual adjacent lobules (Fig. 3).

Pleiotropic Roles for ECM in the Mammary Gland

The ECM provides physical support that is essential foroverall tissue architecture. Changes in ECM properties arecommunicated to epithelial cells via transmembrane recep-tors that provide a conduit between the ECM, thecytoskeleton and its transcriptional machinery [25]. Thus,ECM interactions ultimately determine epithelial cellphenotype. A role for ECM stiffness in the mammarygland has been increasingly explored including the role ofstiffness requirements for establishing apical/basal cellpolarity and lateral cell junctions [26], as well as for cellproliferation [27, 28]. Importantly, Discher and colleagueshave determined that matrix stiffness and mechanotrans-duction are seminal factors in mesenchymal stem celllineage specification [29]. By modulating matrix stiffnessin vitro, this group demonstrated that mesenchymal stemcells acquired a neuronal gene profile on soft matrix, whileacquiring osteoblast-like properties on rigid matrix [29]. Inthe mammary gland, β1 integrin is a cellular “mechano-sensor” that is imperative for maintaining basal progenitorcells, as demonstrated by inability of mammary epitheliumlacking β1 integrin to repopulate the gland upon serialtransplantation [30]. These data suggest that physicalproperties and site-specific composition of adjacent basallamina ECM may contribute to the “stemness” of mammaryprogenitor cells through β1 integrin.

In order to understand how ECM stiffness influences thefunction and fate of mammary epithelial cells, it is vital tounderstand its composition as well as its architecture, whichis largely defined by protein-protein interactions. Whilesecreted ECM proteins can interact through numerous,weak non-covalent bonds, crosslinking these proteins viacovalent chemical bonds is essential for ECM scaffoldorganization, tensional characteristics, and stabilization.Lysyl oxidase (LOX), a copper-dependent enzyme, cata-lyzes intra- and intermolecular crosslinking of fibrillarcollagens and elastic fibers through oxidative deaminationof lysine residues [28, 31, 32]. LOX activity has beenshown to promote tissue stiffness and fibrosis as well asmodulate elastic properties of elastic fibers, establishingLOX as a key regulator of tissue tension [28, 31–33].Tissue transglutaminase 2 (tTG2) has also been implicatedin crosslinking ECM proteins in the mammary stroma. Thecrosslinking activity of tTG2 depends on calcium andproceeds by catalyzing covalent bonds between glutamineand ε-amino groups of lysine residues [34]. Future studiesare needed to examine how LOX and tTG2 are regulated in

Secretoryepithelial cell

ULLC

Lumen

Fibroblast

BL

Figure 1 Electron micrograph image of mammary epithelial cellsfrom a lactating mouse. The solid blue arrow points to the basallamina (BL) adjacent to a putative mammary progenitor cell known asundifferentiated large light cell (ULLC), and a secretory epithelial cell.The dashed red arrow points to intralobular fibrillar collagen adjacentto a fibroblast. Scale bar represents 5 microns

J Mammary Gland Biol Neoplasia (2010) 15:301–318 303

the mammary gland and to determine their uniquecontributions to ECM function.

ECM as a Reservoir and a Source for Growth Factorsand Cytokines

Growth factors and cytokines can bind to ECM proteins,identifying another avenue by which ECM can regulatemammary morphogenesis and function. For example, trans-gluaminase crosslinks the large latent complex of trans-forming growth factor β (TGF-β), known as latent TGF-βbinding protein, to ECM proteins [35]. Growth factors andcytokines bind to ECM through glycosaminoglycans (GAG),which are anionic linear polysaccharides consisting ofrepeating hexuronic acid and hexosamine. GAG can bethought of as “glue” that allows growth factors to “stick” tobasal lamina and fibrillar ECM proteins. Interestingly,different classes of GAGs are associated with distinct aspectsof mammary ductal elongation [20]. Silberstein et al. andWilliams et al. used electron microscopy (EM) to show thatthe basal lamina surrounding the tips of end buds in mice isthin and rich in hyaluronic acid, while the basal lamina alongthe flank of end buds is thicker and associated with sulfated

GAGs [20, 36]. These observations are interesting becausethe terminal end bud tips are actively elongating structureswhile the flank of the end bud is not [37]. These observationsraise questions on how GAG composition contributes toECM thickness, rigidity, and the ability of ECM to serve as areservoir for growth factors and cytokines, as well as theability of epithelial cells to penetrate into the surroundingstroma and proliferate.

Release and activation of growth factors and cytokinesfrom ECM can occur by altering matrix stiffness and/orinducing ECM proteolysis [38, 39]. For example, TGF-βactivity can depend on fibrillar ECM proteolysis or alteredmatrix stiffness. TGF-β is secreted as a large latentcomplex (LLC) by multiple cell types and associates withfibrillar ECM proteins [40]. TGF-β is activated byproteolytic cleavage of the latency-associated peptide(LAP) or by conformational change in LAP throughbinding to the αvβ6 integrin receptor [41, 42]. However,Hinz and colleagues recently published evidence suggestingthat mechanical tension is also involved in the release ofTGF-β1 from the ECM [43]. They suggested that highcontractile activity of myofibroblasts on a stiff matrix maylead to a conformational change in LAP, resulting in TGF-β1 release, while on soft matrix the latent complex stays

H&E

Human Breast Mammary Gland ofSD Rat

Mammary Gland of C3H Mouse

Trichrome

a b c

d e f

}

Figure 2 Histological sections from human breast and rodentmammary gland from nulliparous females. a–c H&E-stained nullip-arous human breast tissue (a), nulliparous SD rat mammary tissue (b),and nulliparous C3H mouse mammary tissue (c). In a, solid arrowhead points to fibrous connective tissue (pink) that is dominant inhuman breast tissue. Arrow in the inset box points to intralobular

stroma. Open arrow heads point to adipocytes, which are abundant inrat and mouse glands. d–f Serial sections stained with Masson’strichrome showing collagen as blue fibers in human nulliparous breasttissue (d), nulliparous SD rat mammary tissue (e), and nulliparousC3H mouse mammary tissue (f). Scale bar represents 100 microns


intact due to lack of mechanical stretch [43]. Additionalsupport for mechanical tension regulating ECM function ispresented in studies demonstrating how fibroblast tractionforces lead to large-scale directional patterning of fibrillarECM proteins such as collagen І [44, 45]. Given that ECMtensional requirements and organization are expected tochange throughout mammary gland development, it islikely that the ability of ECM to serve as a reservoir forgrowth factors and cytokines would be altered accordingly.

During expansion of the ductal network, Daniel andcolleagues postulated that TGF-β1 regulates branchingmorphogenesis by inhibiting ductal growth through directsuppression of epithelial cell proliferation and by stimulat-ing production of collagen I, GAG, and chondroitin sulfatewithin the periductal stroma at puberty [46]. Evidence forthis proposal was obtained by administering TGF-β1directly into the mammary gland of young female mice,which resulted in premature inhibition of ductal elongationconcurrent with aberrant and increased deposition of ECMproteins [46, 47]. Moreover, as discussed above, theseresearchers observed high expression of TGF-β3 in theflank region of end buds, suggesting it may positivelyregulate ECM production at this site [48]. Given the growthsuppressive role of TGF-β during pubertal gland develop-ment, it was surprising that expression of TGF-β2 andTGF-β3 transcripts was elevated during pregnancy [48]. Asmentioned by Robinson et al. [48], this observation is notconsistent with the growth inhibitory function that TGF-β(s) has on mammary epithelial cells during ductalelongation. A later study suggested that TGF-β1 onlyimpedes epithelial cell growth in ducts, but not in alveoli,although the mechanism of this differential effect isunknown [49]. During lactation, the expression of TGF-

β(s) is significantly downregulated [48], which mayprevent TGF-β(s) from negatively regulating expressionof milk proteins such as β-casein [48, 50, 51]. Upon theonset of involution when the gland remodels toward its pre-pregnant state, there is an upregulation of TGF-β tran-scripts, particularly for TGF-β3 [52]. TGF-β signaling mayfurther contribute to the remodeling of the involuting glandby inducing ECM production, upregulating MMP expres-sion, and by recruiting immune cells [53–55]. Takentogether, TGF-β(s) expression and activation are tightlyregulated in mammary gland development where theymediate stromal-epithelial interactions, in part by orches-trating ECM deposition and remodeling.

Instructive cues are also provided to cells from the ECMby the controlled release of protease-cleaved ECM frag-ments, termed ‘matricryptins’ [56, 57]. These fragmentsexhibit altered biological functions compared to intact ECMproteins. For example, fibronectin (FN) fragments initiateapoptosis in epithelial cells [58], while intact FN promotescell adhesion and proliferation [59, 60]. A subclass ofmatricryptins, “matrikines,” are short peptides from ECMproteins that function in a similar manner as cytokines [56,57]. Instructive cues from ECM fragments will be furtherdiscussed below in the context of immune cells and ECMremodeling.

ECM Composition in the Mammary Gland

In order to understand the myriad of functions mediated by theECM, one must have an in-depth understanding of the ECMproteins that contribute to each of the distinct ECMsubcompartments. Our current understanding of ECM com-position is rudimentary and largely derives from studies ofrodent mammary gland tissue (mouse and rat). Until recently,the standard method for identifying ECM proteins was bybiochemical isolation and Edman sequencing and/or byantibody-based detection of candidate proteins. Massspectrometry-based proteomic analysis provides an unbiased,high-throughput comprehensive overview of ECM composi-tion. Unfortunately, the traditional digestion approachesrequired to prepare proteins for proteomic analysis haveproven ineffective for ECM proteins due to their resistance toproteolysis. In the past few years, Liang and others have usedmedia conditioned by mammary epithelial cells in vitro toanalyze their secretomes and have identified peptides origi-nating from ECM proteins such as FN, laminin (LN), and typeIV collagen (collagen IV) [61]. An in-solution digestionapproach for trypsin-resistant ECM proteins was recentlydeveloped by Hansen et al., permitting the identification ofknown and previously unknown ECM proteins within the ratmammary gland [62]. In the next section of this review, wedescribe many of the well-known ECM proteins in the

H&E

Human Breast

Figure 3 H&E-stained nulliparous human breast tissue. This histo-logical section demonstrates how adjacent lobules can exist in verydistinct microenvironments. The arrow points to fibrous intralobularstroma and the arrow head points to adipocyte-rich stroma. Scale barrepresents 100 microns


mammary gland and highlight several ECM proteins notpreviously recognized. We discuss these ECM proteins in thecontext of the three distinct subcompartments depicted inFigs. 1 and 2.

Basal Lamina

The terms basal lamina and basement membrane have beenused interchangeably to refer to the highly specialized ECMdirectly underlying the epithelium. While basement mem-brane refers to the ECM adjacent to the epithelium that isvisible by light microscopy, the basal lamina is a sub-lightmicroscope structure visualized only by EM. The basallamina lies immediately beneath the epithelium and is ahighly organized 20–100 nm thick structure (Fig. 1). Basallamina in the mammary gland is thicker along the flanks ofthe terminal end bud compared to the growing bud tip,which correlates with compositional and possibly function-al differences, as mentioned above [36]. The basal laminaconsists of three layers: the middle electron dense layertermed the lamina densa, and the inner and outer less-denseelectron layers referred to as the lamina lucida externa andinterna, respectively [63]. The lamina lucida layers containhigh levels of proteoglycans (hence they appear as clear or‘lucid’ areas) that decorate a meshwork of small filamentsthat attach the lamina densa to the epithelial cell surface,and to the collagen fibrils within the stroma on the basalside. This creates a highly organized ECM layer betweenthe epithelial cell and its underlying stroma. The majorconstituents of basal lamina are LN, collagen IV, nidogens,and perlecan.

Laminins LNs are large heterotrimeric glycoproteins(~900 kDa) found in at least 15 different combinations thatare derived from five α, three β, and three γ subunits,coded from distinct genes [64]. Each LN chain includesglobular, coiled coil, and rodlike regions [65]. The threedistinct chains are intertwined at the coiled coil regions bydisulfide bonds to form a cross-shape structure [65]. LNdeposition appears to be restricted to the region of the basallamina, is in direct physical contact with epithelial cells,and thus is likely critical mediators of epithelial cell-ECMinteractions in vivo. Different LNs can be associated withspecific tissue compartments or organs. For example, LN8(α4, β1, γ1) is expressed by endothelial cells andcontributes to the vascular basal lamina [15, 66], whileLN10 (α5, β1, γ1) is associated with villus cells of thesmall intestine [67]. These and other examples suggest thatparticular LNs may be coupled with tissue-specific differ-entiation or function. In this review, we will focus onLN111 (α1, β1, γ1; known as LN1) and LN332 (α3, β3,γ2; previously known as LN5) due to their establishedpresence in the mammary gland.

LN111 is one of the major constituents of the basallamina and was the first LN characterized. LN111 wasisolated from a poorly-differentiated murine sarcoma(Engelbreth-Holm-Swarm (EHS) sarcoma), and is a princi-pal component of Matrigel, a commercially-availablebasement membrane substitute [68, 69]. LN111 is neces-sary for cell differentiation as documented in murineembryogenesis, where loss of any LN111 chain (α1, β1,or γ1) causes embryonic lethality due to improper basallamina assembly and function [70, 71]. An insightfulexample was obtained after deletion of specific domainsin the C-terminus of the LN α1 chain [9]. The C-terminusof the LN α1 chain consists of 5 globular domains withknown cell binding motifs [72]. Deletion of the α1 globular4–5 domains, which are binding sites for the ECM receptordystroglycan, impeded pluripotent epiblast differentiation[9]. Interactions between α1 globular 4–5 domains anddystroglycan are crucial for gastrulation and for theformation of the primary germ layers, likely because LNis needed for epithelial cell polarization [9]. Using a 3Dculture model, Bissell and colleagues showed that LN111-rich reconstituted basement membrane is pivotal forapicobasal polarization of mammary epithelial cells andfor lumen formation of acini [73–75]. Cultured luminalmammary epithelial cells did not form acini in collagen Igels unless the cells were mixed with myoepithelial cells[76]. Petersen and colleagues identified myoepithelial cellsas the primary source of LN111 via in vitro assays andthrough staining of human breast tissue [76]. The in vitrostudies have demonstrated that LN111 instructs acinarformation, in part by facilitating deposition of amammary-specific basal lamina, given that newly-synthesized LN332 and collagen IV are incorporated intoa basal lamina-like structure in a 3D culture model in thepresence of LN111 [77, 78].

LN332 is expressed by mammary epithelial cells and caninduce adhesive contacts in epithelial cells via α6β4 orα3β1 integrins [79, 80]. Moreover, MMP2-cleaved LN332fragments (from the γ2 chain) have been shown to bind theepidermal growth factor receptor (EGFR) [81]. LN332fragment-EGFR interaction was associated with pro-migratory effects on mammary epithelial cells as well asdownstream activation of EGFR signaling, although theresults from the cell growth assay were inconclusive [81].Further, by culturing freshly-isolated murine mammaryepithelial organoids that included luminal epithelial andmyoepithelial cells in a 3D culture model, Werb andcolleagues demonstrated that the front edges of actively-elongating ducts were devoid of intact LN332 [80].Moreover, LN332 fragments are present in the mammarygland during mid-pregnancy and involution, suggesting thatLN332 contributes to the tissue remodeling that occursduring these stages [81, 82]. Using a pan anti-LN antibody,


LN fragments were apparent in mammary tissue duringearly involution in the rat [23]. Nevertheless, the functionalconsequences of LN turnover during involution are stillunknown. While LNs experience turnover during earlyinvolution of the gland [23], LN transcript and total proteinlevels change only modestly with pregnancy, lactation andinvolution, suggesting a fundamental requirement for LNsin mammary epithelial maintenance, rather than for stage-specific functions.

Collagen IV Collagen IV is also a major component of thebasal lamina and is thought to be its primary scaffoldprotein [16, 83]. Unlike fibrillar collagen, collagen IV is anetwork-forming collagen. It is a heterotrimer and can becomprised from six possible genetically distinct α chains.Collagen IV α chains trimerize to form three distinct‘protomers’ that can be distributed in a tissue-specificmanner [84]. Two collagen IV protomers bind through theC-terminal non-collagenous 1 (NC1) domain to form ahexamer [16]. Hexamers interact with one another throughN-terminal 7 S domains to generate a tetramer [16].Yurchenco and colleagues demonstrated that the lateralinteractions among tetramers created a polygonal networkof collagen IV and a 3D mesh [16, 85]. The α1.α1.α2protomer was suggested to form the initial sheet-like basallamina during embryogenesis [84]. Disruption of the COL4α1 or α2 locus in mice is embryonic lethal [84].Surprisingly, in collagen IV-deficient mice, LN111 andnidogen 1 were deposited and organized into a basallamina-like structure, during early embryogenesis (prior toE10.5). The basal lamina in these mice had a discontinuousand altered structure, suggesting that collagen IV is notrequired for LN111 and nidogen assembly but is required tostabilize the nascent basal lamina [84]. Furthermore, othercollagen IV α chains did not compensate for loss of theα1.α1.α2 protomer [84]. Later in development, theα1.α1.α2 protomer is replaced with different collagen IVprotomers such as α3.α4.α5 in the glomerular basal lamina[86]. Evidence for the importance of protomer tissuespecificity has been obtained from patients with Alportssyndrome who have a mutation in the COLα5 chain. Thismutation leads to absence of the α3.α4.α5 protomer (IV)based network in the glomerular basal lamina, impeding therequired switch from the fetal α1.α1.α2 protomer to theα3.α4.α5 protomer (IV) [86]. Alports syndrome leads toprogressive renal failure among other conditions [87].

Although it is unclear which α chains comprise collagenIV in the mammary gland, collagen IV deposition has beenfound necessary for proper formation of the mammarylamina densa. Disruption of collagen deposition in the basallamina in vivo resulted in collapsed alveolar structures inproliferative mammary epithelium, resulting in a phenotypesimilar to that seen during early involution [88]. In another

study, Kidwell and colleagues demonstrated that mammaryepithelial cells preferentially attached to collagen IV versescollagen I in vitro [89]. Hence, collagen IV is essential forsupporting basal lamina structure and provides an anchorfor mammary epithelial cells, indicating its specific role inmaintaining cell viability. On a different note, crypticdomains of type IV collagen fragments were demonstratedto function as anti-angiogenic factors. Tumstatin is afragment from the C-terminus of collagen IV α3 chainand inhibits angiogenesis by blocking αVβ3 integrinsignaling [90, 91]. Further, during early to mid-involution,alveolar loss is associated with both basal lamina remodel-ing and a decrease in vascular support needed for lactation[90, 92]. Thus, there may be a link between basal laminaturnover and vascular regression that occurs during early tomid-involution.

Nidogen Nidogens (also known as entactins) are sulfatedglycoproteins (150 kDa) synthesized by fibroblasts as invarious tissues such as skin, lung, central nervous system, andlimb [93–95]. There are two genes in the nidogen family thatencode nidogen 1 and nidogen 2. Nidogens are incorporatedinto the basal lamina upon secretion [93, 96]. Germ layercooperation in the assembly of basal lamina is highlighted bythe fact that mesodermal-derived fibroblasts make nidogenand ectoderm/endoderm-derived epithelial cells produceLNs. Nidogen is found in a 1:1 stoichiometric ratio withLN and can be viewed as a stabilizing component of thebasal lamina by serving as a bridge between LN111 andcollagen IV [97]. For example, the globular domain G3 ofnidogen 1 binds to the C-terminal short arm of a LN γ1chain and the G2 domain of nidogen binds collagen IV [98,99]. Nevertheless, deletion of nidogen 1 in mice demonstrat-ed that it was not essential for basal lamina assembly [100],as nidogen 2 appeared to compensate [101]. Furthermore,mice lacking both nidogen 1 and nidogen 2 do not die duringembryogenesis, indicating that nidogens are not necessaryfor basal lamina assembly in most organs. However, lungsfrom newborn mice deficient in both nidogens had smallalveolar spaces, thicker septa, and a prenatal decrease inexpression of basal lamina constituents such as LN γ1 chain,which recovered to control levels by the perinatal period[102]. In addition, the ability of type II pneumocytes toproduce pulmonary surfactant may be impaired given thatthe expression of surfactant protein B was reduced in doubleknockout newborn mice [102]. Pulmonary surfactant isimportant in lowering the alveolar surface tension as wellas maintaining alveolar structure. Taken together, these datasuggest that nidogen 1 and nidogen 2 are needed for properfunction of alveolar epithelial cells in the lung. Their absencein the lung may be the reason mice lacking both nidogensdie shortly after birth [102]. Nidogens also support tissue-specific differentiation as nidogen 1 promotes the ability of


LN111 to induce β-casein expression by mammary epithelialcells in vitro [103]. Collectively, these and other data suggesta functional relevance for nidogens in a tissue-specificmanner, but surprisingly, also indicate that nidogens are notessential for basal lamina formation during embryogenesis.

Perlecan Perlecan is a proteoglycan that consists of a470 kDa core protein with covalently-linked heparansulfate, a specific-class of GAG, which increases themolecular weight (MW) of perlecan to over 800 kDa[104]. Analysis of perlecan distribution during developmentrevealed its expression in the basal lamina of liver, lung, thevascular system, and other tissues as well as in interstitialstroma such as the interterritorial ECM of articular cartilage[105]. Further, mammary epithelial cells have been shownto secrete heparan sulfate proteoglycan, suggesting epithe-lial cells as a possible source of perlecan [17]. Disruption ofthe gene encoding perlecan (Hspg2) caused embryoniclethality in a large portion of mice. Lethality occurs atE10.5 due to defective cephalic development and effusionof blood to the pericardium, which is thought to result frombasal lamina deterioration [106, 107]. The remainingperlecan-null mice died perinatally with severe skeletalanomalies, presumably due to the role of perlecan in bonematrix (as described above) [106, 107]. Moreover, Fässlerand colleagues observed discontinuous areas in the basallamina in vivo that separated the neuroepithelium from theunderlying mesenchyme [106]. All together, the phenotypesof the null mice suggest that perlecan is essential for basallamina assembly in several organs and for bone matrixfunction. Additionally, perlecan’s soluble C- terminalfragment, endorepellin, disrupted capillary morphogenesisin vitro by inhibiting endothelial cell migration, andangiogenesis [108]. Moreover, perlecan mediates the effectsof several fibroblast growth factor (FGF) family members.Perlecan was suggested to interact with FGF2 through itscore protein and GAG group [109]. These interactions canenhance the potency of FGF2 signaling through FGFR1and FGFR3 in vitro and consequently have a pro-angiogenic effect [109, 110]. As with most ECM proteins,perlecan provides instructive cues dependent on contextand structural integrity.

Intra- and Interlobular Stroma

Immediately adjacent to the basal lamina are 10–100 μmthick bands of organized, collagen-rich stroma, known asintralobular stroma, that surround individual alveoli(Fig. 2a, arrow in the insert box). Clusters of alveoli thatcompose a single lobule are further surrounded by bands ofstroma referred to as interlobular stroma. Fibrillar collagensare the dominant structural constituents, with several other

ECM dictating intra- and interlobular stroma organizationand functions.

Fibrillar Collagens Collagen I, III and V are the majorfiber-forming collagens, where their individual proteinsorganize into bundles of various thicknesses and lengths toform the pink or blue fibrous material observed in H&E(Fig. 2a–c) and trichrome-stained (Fig. 2d–f) mammarysections, respectively. Collagen І has been demonstrated tobe synthesized by fibroblasts, chondroblasts, osteoclasts,and odontoblasts during normal development and byactivated fibroblasts during wound healing. Thus, mesen-chymal cells are thought to be the primary source ofcollagen І. The collagen I precursor, procollagen (500–600 kDa) consists of two identical α1 (I)-chains and one α2(I)-chain, each derived from a unique gene. Each α chain ismade up of approximately 300 Gly-X-Y repeats where X isfrequently proline, and Y is hydroxyproline [32, 111]. TheGly-X-Y repeats are essential for ‘self-assembly’ ofcollagen chains into a triple helix procollagen structure,where all the glycines face inward while the larger aminoacids such as proline cover the outer positions [111, 112].After assembly into the triple helix, peptidases remove non-helical registration peptides from the secreted procollagen[111] and subsequently it is transformed into insoluble300 nm tropocollagen [111]. At the end of each triple helixtropocollagen molecule there are short non-helical telopep-tides. These allow further stabilization and aggregation bycovalent intra- and intermolecular crosslinking via LOXresulting in the basic fibrillar structure of collagen observedin histological sections [32, 111]. Based on its ubiquitousand abundant presence, fibrillar collagen І is considered thebackbone of stroma, including that in the mammary gland.As such, the physical and biochemical properties ofcollagen І likely contribute significantly to the functionalattributes of the mammary stroma.

Work from the Taylor-Papadimitriou and Sonnenscheinlabs demonstrated that collagen I supported mammaryductal formation in 3D culture [113, 114]. Further, whenthe collagen-specific α2 integrin subunit was knocked outin vivo, branching complexity in mammary glands wasreduced [115]. Nonetheless, the epithelial layer in vivo isseparated from the intralobular stromal ECM by the basallamina. Therefore, how do these ECM subcompartmentsand stromal cells such as fibroblasts cooperate to influencemammary epithelial morphogenesis? Krause et al., embed-ded mammary epithelial cells with and without fibroblastsin collagen I gels and in Matrigel-collagen I gels [114].Epithelial cells alone could only form ductal structures incollagen I gels in comparison to Matrigel-collagen I gels,however when co-cultured with fibroblasts, the epithelialcells formed ductal structures in both gel conditions [114].Moreover, fibroblast-secreted factors such as hepatocyte


growth factor (HGF) can enhance the ability of mammaryepithelial cells to form duct-like structures in collagen І gels[113]. Collectively, these studies demonstrate that inter-plays between fibrillar collagens, integrins, and growthfactors are required for mammary duct development.

Fibrillar collagen-epithelial cell interactions are alsocritical for supporting the mammary epithelium duringpregnancy and lactation. Collagen-associated receptor,DDR1 may control the tremendous increase in epithelialproliferation that occurs during pregnancy [8]. DDR1-nullmice demonstartaed hyperproliferation of the mammaryepithelium during pregnancy and were unable to lactate,despite the ability of luminal epithelial cells to expressnormal transcriptional levels of milk proteins [8]. Hence,interrupting the collagen-DDR1 interaction causes misguid-ed instructive cues that are imperative for proper alveolarexpansion and secretory function [8].

With cessation of milk secretion and weaning, themammary epithelium involutes and returns toward itspre-pregnant, non-secretory state. The stroma in theinvoluting gland shares attributes with fibrotic, desmo-plastic stroma seen during wound healing. Duringinvolution, fibrillar collagens are upregulated in themammary gland and an influx of innate immune cellssuch as macrophages are observed [116–118]. Based ongene array data, the onset of involution is characterized byelevated expression of several fibrillar collagens includingcollagen І, III, and V as well as an increase in theexpression of genes involved in ECM turnover [23, 52].We and others have demonstrated that non-fibrillardenatured collagen can be a positive chemoattractant formacrophages in vitro [118]. Although it is unclear whethermacrophages are necessary for collagen accumulation orremodeling of the mammary ECM during involution,monocyte chemoattractant protein 1 (MCP-1) increasesin early to mid-involution [118]. The presence of MCP-1was shown to be important for collagen fiber formation ina chemical-induced skin fibrosis model, as MCP-1 nullmice were resistant to chemical-induced skin fibrosis,including fibrillar collagen deposition [119]. Further, theloss of MCP-1 was associated with a decrease in macro-phages and other innate immune cells, in addition to anincrease in the small leucine-rich proteoglycan (SLRP)decorin. The relationship between SLRP and collagenfibrillogenesis will be discussed later in this review.Collectively, the data suggest that fibrillar collagen-macrophage interactions may, in part, drive the tissueremodeling programs within the involuting mammarygland.

Another open question is whether unique functions areimparted by distinct fibrillar collagens with respect to collagenbundle formation, ECM turnover, and cell-collagen interac-tions. Although collagens III and V form oriented super-

structures characteristic of fibrillar collagen, these collagenshave features distinct from collagen I. For example, localizedhelix instability in collagen III can be caused by the highercontent of glycine in α1 (III) chains [32]. This structuralfeature may cause an increase in the turnover rate of collagenIII and subsequently the collagen III matricryptins may serveas potent chemoattractants for innate immune cells. Further-more, collagen III is a homotrimer consisting of a single αchain, while collagen V is a heterotrimer composed of threedifferent α chains [111]. These variations in primarystructure likely cause additional changes in biochemicalproperties, as fibrillar collagen I, III, and V interact with oneanother to assemble into supramolecular collagen bundles[111]. The assembly of these bundles is further supported byshort triple-helical fibril associated collagens with interruptedtriple helices (FACIT) collagens such as collagen ІX, whichcan function as crosslinking bridges [32, 111]. Interactionsbetween fibrillar collagens and FACIT collagens may beimportant for mammary gland remodeling by dictatingcollagen organization and turnover.

In summary, the function and expression levels offibrillar collagens are altered during the various stages ofmammary gland development. These studies suggestunique roles during duct formation, alveolar expansionduring pregnancy, as well as to the demoplastic-likemicroenvironment during involution, highlighting the dy-namic role of fibrillar collagens in the mammary gland.However, even for ECM proteins as ubiquitous as fibrillarcollagens, our basic understanding of both function andmechanism of action remains largely unknown.

Fibronectin FN is a large dimeric glycoprotein (~500 kDa)that mediates cell adhesion, migration, proliferation, andbranching morphogenesis. FN is encoded by a highly-conserved single gene and its deletion causes embryoniclethality during gastrulation, emphasizing its essentialfunction [120]. FN consists of three distinct repeatingmodules known as type І, II, and III domains [121].Although FN protein is encoded by a single gene, there areseveral variant FN forms that arise due to alternativesplicing [121, 122]. Specific examples are the spliced sitesin the type III domain, termed extra domain (ED) EDA,EDB, and IIICS. Extracellular stimuli such as HGF havebeen shown to regulate splicing in vitro [122]. EDA andEDB have been implicated in wound healing, as theirexpression is undetectable in normal liver but is rapidlyupregulated during injury [123]. In addition to its essentialrole in early embryogenesis, FN is an essential regulatoryprotein for salivary gland branching morphogenesis [124].Yamada and colleagues blocked cleft formation andbranching in the salivary gland ex vivo by downregulatingFN expression in salivary epithelial cells via siRNA or byinhibiting FN-epithelial cell interaction via anti-FN anti-


body or antibodies against β1 integrin subunit [124].Further, branching morphogenesis was rescued by addingexogenous FN to siRNA-treated glands [124]. Thesestudies have clearly demonstrated that FN is required forsalivary gland branching morphogenesis, although, thequestion of whether this requirement is FN variant-specific is unknown [124].

Ovarian steroids are suggested to regulate FN expressionin the mammary gland [24]. Haslam and colleaguesmeasured a three-fold increase in FN protein levels aroundmammary ducts between pre-puberty and sexual maturity[24]. Moreover, ovariectomy of sexually-mature miceresulted in a 70% decrease in the FN concentration in themammary epithelium [24]. FN and the fibronectin receptorα5β1 integrin are dramatically downregulated during late-pregnancy and lactation, and then upregulated duringmid-involution [23, 24]. Interestingly, the EDA isoformassociated with pathological conditions in the liver isdevelopmentally regulated in the rat mammary glandsimilar to total FN, suggesting a role for EDA within thenormal mammary gland [23]. During the early stages ofinvolution, elevated matrix metalloproteinase (MMP) ac-tivity correlated with higher levels of cleaved FN [23].Additionally, FN fragments can induce MMP9 activity inmammary epithelial cells in vitro, suggesting a positivefeedback loop between FN fragments and MMP activity[23]. This has implications for clearance of the secretoryepithelium in involuting glands because FN fragments caninduce apoptosis in epithelial cells [58].

Tenascins The tenascin (TN) family consists of 5 members:TN-C, TN-R, TN-W, TN-X, and TN-Y. TN-C was the firstmember of the TN family to be discovered, and is a six-armed glycoprotein with a 3D structure known as hexab-rachion. Each arm contains N-terminal structural domainsfor self-assembly, followed by EGF-like repeats,alternative-spliced FN type III domains, and a C-terminalglobular fibrinogen-homology domain [125]. The MW ofTNs ranges between 180 and 300 kDa, which may dependon alternative splicing [125, 126]. TN is suggested todisplay anti-adhesive properties via disruption of theinteractions between the transmembrane heparan sulfatereceptor sydecan-4, α5β1 integrin receptor, and fibronectin[127, 128]. An anti-adhesive effect of TN-C on cells suchas satellite cells has been shown to be context-dependentbecause other cells, for example, embryonic dorsal rootganglion explants, were able to spread, migrate, and growon TN-C ex vivo [129]. Disruption of the TN-C gene inmice did not produce any visible abnormalities [130].Although, Kusakabe and colleagues demonstrated neuro-logical defects in TN-C null mice [131, 132]. Nonetheless,TN-C expression is associated with multiple tissues duringembryogenesis and pathological conditions. In general, TN-

C expression is transient and largely restricted to embryo-genesis, yet it is upregulated in breast cancer [125, 133].Hence, TN-C has been described as an oncofetal protein.TN-C was also demonstrated to be overexpressed in skinduring wound healing, particularly in the dermis–epithelialjunction [134]. In the mammary gland, TN-C is transientlyexpressed in dense stroma surrounding the buddingepithelium during rodent embryogenesis, with little to noexpression observed in the quiescent adult gland [135]. TN-C expression is again upregulated during lactation and earlypostpartum involution [23, 136]. TN-C was found both inthe milk of lactating mice and women [137, 138]. Thefunction of TN during lactation is unclear, especially inlight of fact that β-casein expression was inhibited inmammary epithelial cells cultured on reconstituted base-ment membrane containing exogenous TN-C [136].

Another TN family member that was identified in themammary gland is TN-X [62]. TN-X has only atribrachion structure and is prominently expressed in testisand muscle [139]. Burch and colleagues identified aconnective tissue defect in TN-X-deficient patients thatinvolved hyperextensible skin and joints, vascular fragil-ity, and improper wound healing similar to Ehlers-Danlossyndrome [140]. These observations suggest that TN-Xmaintains tissue elasticity. During lactation the mammaryepithelial cells can experience considerable stretch forces,and it is of interest to determine whether TN-X providessuch a function at this time.

SPARC SPARC (secreted protein acidic and rich in cyste-ine, also known as osteonectin) has been identified in themammary gland by gene array and mass spectrometrybased proteomics [52, 62]. SPARC is a small, 32 kDasecreted glycoprotein that mediates cell-matrix interactions[141]. SPARC contains a calcium-binding C-terminalextracellular module, follistatin-like module, and N-terminal acidic module [142, 143]. SPARC-null miceexhibit abnormal collagen fibril morphology [144]. Usingpicrosirius red staining, the effect of SPARC on collagenfibrillogenesis was evaluated in the dermis [144]. Com-pared to wild type mice, the dermis in SPARC-null micecontained less striated collagen fibers [144]. Sage andcolleagues found that SPARC preferentially binds to ECMproteins such as collagen I and LN111, but not to collagenIII [145]. They also demonstrated that SPARC displays anepitope-specific anti-adhesive function that causes cellrounding [145]. In contrast, SPARC was shown topositively regulate FN assembly and integrin-linked kinaseactivity, which resulted in FN-induced stress fiber forma-tion in lung fibroblasts [146]. SPARC has been reported tobe developmentally upregulated within the mammary glandduring the transition between lactation and postpartuminvolution, correlating with increased levels of collagens


and FN [23, 118]. Future research focused on SPARCfunction in collagen fibrillogenesis and FN assembly in adynamic organ such as the mammary gland is warranted.

Small Leucine-Rich Proteoglycan (SLRP)

SLRP family members are characterized by N-terminalcysteine-rich motifs and tandem leucine-rich repeats (LRR)in their core protein. The core protein of SLRP is‘decorated’ with one or more GAG chains. The SLRP genefamily was categorized into five different classes based onseveral factors such as gene homology, distinctive N-terminal cyteine-rich motifs, and characteristic GAG sidechains [147]. SLRPs were initially thought to only providestructural support and participate in collagen fiber forma-tion. However, additional biological functions for SLRPswere recently identified [147]. SLRPs can directly bind tocell surface receptors and to growth factors, implicatingthem in various signal transduction pathways and cellularprocesses [147]. We will focus below on decorin andbiglycan from class І, as these SLRPs have been deter-mined to be present in the mammary gland via gene arrayand proteomics in multiple studies [52, 62].

Decorin Decorin core protein (~38 kDa) is covalentlylinked with a single chondroitin sulfate (CS) or dermatansulfate (DS) chain resulting in an overall MW of 90-140 kDa [148]. Decorin is a secreted protein and isexpressed in various cell types such as fibroblasts andastrocytes [149, 150]. Although decorin-deficient miceexperienced skin and lung fragility, possibly due toaberrant organization of fibrillar collagen, they are viable.Variability in the shape and size of collagen fibrils in thesemice was observed compared to wild-type mice, asdetermined by EM of dermal collagen [151]. Based onthese studies, decorin has been suggested to be pivotal forproper spatial alignment of stromal collagen fibers.Another function of decorin was demonstrated by Iozzoand colleagues, where decorin suppressed signalingthrough ErbB2 in breast cancer cells in vitro and inxenograft models [152]. They suggested that decorinmay inhibit EGFR signaling by initiating EGFR internal-ization and degradation by caveolar endocytosis [153].Angiogenesis was also negatively affected by decorin as itdownregulated vascular endothelial growth factor expres-sion in tumor cells [154]. When endothelial cells werecultured in media from decorin-expressing tumor cells,they failed to migrate or form capillary-like structures invitro [154]. An additional role of decorin is to bind TGF-β1 and sequester its activity in vitro [155]. Given thatdecorin is crucial for proper fibrillar collagen organization,one question concerns the role of decorin in mediatingtensional changes required for TGF-β1 activation as

discussed in previous sections [26]. Array data demon-strate that decorin is upregulated during mammary glandinvolution [52], where it likely contributes to the transientincrease in collagen fibers [118].

Biglycan Biglycan is also a member of the SLRP class Іfamily and is comprised of a core protein (~38 kDa) thatis covalently attached to two GAG chains (chondroitinsulfate and/or dermatan sulfate) with an overall MW of150–240 kDa [148]. Young and colleagues observed areduction in bone formation and mass in biglycan-deficientmice [156]. Disruption of biglycan gene expression didnot cause upregulation of decorin expression and vice versa[151, 156, 157], although both biglycan and decorin belongto the same SLRP subfamily [157]. These results suggestthat decorin and biglycan have distinct, non-overlappingroles [156]. Schaefer et al. have tested the function ofbiglycan in maintaining renal tissue elasticity underpressure-induced injury caused by unilateral ureteralligation [158]. They concluded that biglycan can induceelastic fiber-associated protein fibrillin 1 expression inrenal fibroblasts and mesangial cells, which subsequentlyhelp to retain elastic properties of the damaged tissue[158]. Their data is especially intriguing when it istaken together with work from Salgado et al. whodemonstrated dynamic expression and localization ofdecorin and biglycan in the mouse uterus during the estrouscycle and pregnancy, suggesting that it is hormonally-regulated [159, 160]. These studies raise the question ofwhether biglycan and decorin are also hormonally-regulated in the mammary gland and whether theyinfluence the elastic properties of the gland during periodsof expansion. Moreover, biglycan transcript abundance inthe mammary gland increased four- to five-fold duringthe transition from lactation to involution [52, 118].

Importantly, several members of the class II SLRPfamily including lumican, mimecan, and prolargin havebeen identified in the mammary gland, as well asevidence for their differential expression across thereproductive cycle [52]. Future studies are required toinvestigate the function of the class II SLRP family in themammary gland.

Fibrous Connective Tissue

The third structurally-distinct stroma in the mammarygland is the relatively acellular fibrous connective tissue,which is often found in large swaths (mm to cm range inthe rat and human) and is characterized by the absenceof epithelium and presence of fibroblasts, immune cells,and high fibrillar collagen content (Fig. 2a, solid arrowhead). While the fibrous connective tissue is considered tobe separate from the intra- and interlobular stroma, these


compartments share many of the same ECM proteins suchas fibrillar collagens, FN, TN, SLRPs, and SPARC.However, a unique feature of fibrous connective tissue isthe presence of elastic fibers.

Elastic Fibers Elastic fibers provide structural support andelasticity to various tissues. Elastic fibers are comprised ofa 31 kDa secreted protein called microfibril-associatedglycoprotein (MAGP) and another glycoprotein, the350 kDa fibrillin protein that assembles into 10- to 12-nmmicrofibrils [31]. Microfibrils then associate with otherelastic fiber constituents including elastin, fibulins, andproteoglycans. The process of organizing elastic fibers inthe connective tissue begins with the formation of theMAGP/fibrillin microfibrils that align parallel to fibroblastsand serve as tracks for the deposition of tropoelastin(70 kDa), which is a soluble precursor of elastin.Tropoelastin is rich in hydrophobic amino acids andcontains low amount of polar amino acids including severallysine derivatives. The lysine derivatives are imperative forcovalent crosslinking between monomeric elastin via LOX[31]. Microfibrils are described as a scaffold that facilitatesthe alignment of soluble monomeric elastin to coalesce andorganize into amorphous elastin [31]. As elastic fibersmature postnatally, elastin becomes the dominant compo-nent, with microfibrils being gradually displaced to theouter layer. The resultant fibers have physical propertiesthat can withstand changes in the mechanical environment[161]. An additional constituent of elastic fibers is thefibulin family (50–200 kDa), which have calcium-bindingsites and consist of І, II, III domains with a central segmentthat is composed of EGF-like modules. Fibulin 5 (66 kDa)displays strong calcium-dependent binding to tropoelastin,while it weakly binds to carboxyl-terminal of fibrillin 1[162]. Further, fibulin 5 knockout mice were generated toanalyze fibulin 5 function in elastic fiber formation in thedermis. These mice demonstrated a requirement for fibulin5 in tropoelastin deposition and potentially for optimalLOX activity in vivo [162, 163]. Moreover, proteoglycanssuch as biglycan and decorin were implicated in elasto-genesis by binding to tropoelastin, fibrillin 1, and MAGP[158, 164, 165]. While much is known about the functionsof elastic fibers in other systems such as lung, skin, andvascular system, their function in the mammary gland isstill elusive. Elastic fibers are anticipated to be particularlyimportant in the lactating mammary gland as the tensileforces continuously change due to suckling, milk accumu-lation between nursing, and myoepithelial contraction onthe secretory alveolar epithelial cells. Further research onthe function and regulation of the fibrous connective tissue,including elastic fibers, is necessary given the correlationsbetween higher mammographic density, tissue tension, andincreased breast cancer risk [28, 166, 167].

Future Directions

Immune Cells and ECM Remodeling

The ECM of the mammary gland plays a role in theinfiltration and activation of immune cells. We havepreviously characterized involution as a wound healingmicroenvironment with high levels of alternatively activat-ed or M2 type macrophages [118]. How the immune cellsare recruited and ‘activated’ still remains unclear. However,many of the proteins that make up the ECM can fragmentinto matrikines and matricryptins, which have been dem-onstrated to influence leukocyte infiltration in othersystems. Fragments of collagen I, collagen IV, LN andnidogen-1 have all been shown to promote chemotaxis ofmonocytes and neutrophils within the interstitial tissue, butit remains unknown whether tissue-derived matrix frag-ments enter the blood stream to recruit immune cells oraugment their motility upon arrival to the local tissue [118,168–171]. Once in the mammary gland, macrophages andneutrophils secrete proteases including MMP9 and elastasethat are able to breakdown the ECM [118, 172, 173].Without the influx of macrophages or neutrophils and theircrucial proteases, remodeling of the mammary tissue to itsnon-secretory postpartum state could potentially be delayedor incomplete. Werb and colleagues have evaluated theimportance of secreted proteases during involution in themammary gland by inhibiting a serine protease, plasmakallikrein. They demonstrated that inhibition of this mastcell-associated protease during involution caused an accu-mulation of fibrillar collagen and delayed repopulation ofadipocytes, thus preventing the gland from returning to itspre-pregnant state [174]. Combined, these studies indicatethat ECM turnover and immune cell function are highlylinked, creating a feedback loop that is necessary tocomplete gland remodeling after lactation.

In addition to facilitating immune cell infiltration, ECMfragments may act as ligands to receptors present onleukocytes in the mammary gland. Fragments of biglycan,heparan sulfate, and hyaluronan have been shown to act asligands for toll like receptor 4 (TLR4). TLRs are part of thepattern recognition receptor family expressed on the cellsurface of innate immune cells and dendritic cells. When aligand binds to a toll like receptor the immune cell becomesactivated and/or secretes cytokines that can further activatecells of the adaptive immune system. Babelova et al. recentlydemonstrated that soluble biglycan binds to the TLR 2/4 onmacrophages, stimulating them to synthesize and releaseinterleukin-1β, a proinflammatory cytokine [175]. Addition-ally, heparan sulfate and hyaluronan have been shown tobind to the TLR4 on dendritic cells, resulting in dendritic cellmaturation [176, 177]. Once mature, dendritic cells are ableto activate the adaptive immune system, where adaptive


immune cells respond and migrate to the site of ECMremodeling. Consistent with a similar mechanism of adaptiveimmune cell activation due to ECM fragmentation in themammary gland, the presence of B cells in the mammarygland during involution has been confirmed; however theirrole remains elusive [172]. The adaptive immune system istypically activated after the innate response, although B cellshave been shown to infiltrate the gland during early to mid-involution, prior to the peak in macrophage recruitment[172]. The presence of B cells may indicate that cells of theadaptive immune system can be attracted to the gland,similar to the innate immune cells, by the presence ofproteolytic ECM fragments. Interleukin-4 and fibronectin,which are both upregulated during involution, have beenshown to stimulate motility of B cells in vitro, and mayaccount for the increased presence of B cells in themammary gland during involution [118, 178]. The infiltra-tion of immune cells such as macrophages, neutrophils, mastcells, and B cells into the postpartum mammary gland duringinvolution suggests both the innate and adaptive immunesystems play an important role in returning the gland towardits pre-pregnant state. However, with limited research on thefunction of immune cells in the mammary gland, additionalstudies are necessary to determine the role of the ECM inimmune cell activation, as well as the role for immune cellsin restructuring the mammary gland in vivo.

Summary

The in situ analysis of mammary stroma by EM, cytologicalstains, and immunohistochemistry have provided snapshotviews depicting the mammary stroma as diverse and highlycomplex. Cell culture studies using purified ECM compo-nents clearly demonstrate distinct, non-overlapping rolesfor ECM proteins with functions that range from inductionof cell death to induction of a secretory phenotype. Studiesusing ECM preparations derived from tissues [EHS tumorand rat mammary glands] further demonstrate the complexand dynamic nature of the endogenous ECM. To further ourunderstanding of the composition and function of ECM inthe mammary gland, it is necessary to increase our ability toobtain better qualitative and quantitative ECM proteomes.Progress in proteomics is beginning to permit non-biased,high throughput, quantitative assessments of ECM pro-teomes. Such results will likely transform future in vitroexperiments by permitting a systems approach to the studyof mammary ECM-epithelial cell interactions.

Acknowledgment We thank Jenean O’Brien and Jaime Fornetti forcritical review of the manuscript. We also thank Dr. Gilbert H. Smith(National Institutes of Health, MD) for providing us with the electronmicrograph image in figure 1.

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